It is recognized that two or more dyes of varying proportions could be used to increase the permutation number of unique combinations of dyes in a single particle. The unique emission wavelengths and fluorescence intensities could be useful for multiparameter or multiplex analysis of a plurality of analytes in the same sample.
Three methods of making colored, fluorescent beads have been disclosed, including: (a) covalent attachment of dyes onto the surface of the particle (e.g. U.S. Pat. No. 4,774,189 Schwartz; U.S. Pat. No. 5,194,300 Cheung), (b) internal incorporation of dyes during particle polymerization (e.g. U.S. Pat. No. 5,073,498 Schwartz; U.S. Pat. No. 4,717,655 Fulwyler), and (c) dyeing after the particle has been already polymerized (e.g. L. B. Bangs, Uniform Latex J Particles; Seragen Diagnostics Inc. 1984).
U.S. Pat. No. 5,194,300 Cheung and U.S. Pat. No. 4,774,189 Schwartz disclose fluorescent microspheres that are coated by covalently attaching either one or a plurality of fluorescent dyes to their surface. However, the features of the particles do not meet the specifications required for multiplex analysis: In U.S. Pat. No. 4,774,189 Schwartz fluorescent proteins are coupled to particles to generate standards for flow cytometric analysis using similar fluorescent proteins. It discloses that particles with different intensities of fluorescence can be generated by covalently attaching fluorescent proteins to particles. However, since these particles were designed for standardization purposes, no attempts were made to couple two fluorochromes simultaneously to the particles. Furthermore, no attempts were made to attach a capture reagent molecule to the same particles. The fluorescent probes used are proteins that denature in harsh conditions such as those used for hybridization of DNA in buffers used for immunoprecipitation that contain denaturing detergents such as SDS. Furthermore, attachment of fluorescent proteins to particles will compromise the binding of capture reagents such as antibodies to the same particles. Their method is therefore not applicable for generating multicolored particles for multiplex analysis. U.S. Pat. No. 5,194,300 Cheung reports small (300 angstrom) fluorescent particle that could be used to enhance signals for detection. The inventors show that it was possible to generate particles that have a single fluorescence intensity and a capture reagent bound to their surface. It was not reported whether binding of the dyes interfere with subsequent binding of biomolecules. Moreover, no attempts are made to generate particles with several different intensities of fluorescence or to couple two colors to the same particle.
The second approach to particle dying is represented by U.S. Pat. No. 5,073,498 Schwartz and U.S. Pat. No. 4,717,655 Fulwyler. The former discloses two or more fluorescent dyes added during polymerization process and randomly dispersed within the body of the particle. However, when such particles are exposed to a single excitation wavelength only one fluorescent signal is observed at a time and thus these particles are not useful for multiparameter analysis especially in a flow cytometry apparatus with a single excitation light source. U.S. Pat. No. 4,717,655 Fulwyler discloses two dyes mixed at five different ratios and copolymerized into a particle. Although five populations of beads were claimed as being obtainable, the fluorescent properties of these beads are not provided. In conclusion, both U.S. Pat. No. 5,073,498 Schwartz and U.S. Pat. No. 4,717,655 Fulwyler represent complex and costly methods for producing multicolored particles comprising internal incorporation of dyes.
The principle of the third method, i.e., internally embedding or diffusing a dye after a particle has been already polymerized was originally described by L. B. Bangs (Uniform Latex J Particles; Seragen Diagnostics Inc. 1984, p. 40) and consists of adding an oil-soluble or hydrophobic dye to stirred microparticles and post-incubation washing off the dye. The microspheres used in this method are hydrophobic by nature. This allows adopting the phenomenon of swelling of such particles in a hydrophobic solvent, which may also contain hydrophobic fluorescent dyes. Once swollen, such particles will absorb dyes present in the solvent mixture in a manner reminiscent to water absorption by a sponge. The level and extent of swelling is controlled by incubation time, the quantity of cross-linking agent preventing particle from disintegration, and the nature and amount of solvent(s). By varying these parameters one may diffuse a dye throughout particle or obtain fluorescent dye-containing layers or spherical zones of desired size and shape. Removing the solvent terminates the staining process. Microparticles stained in this manner will not “bleed” the dye in aqueous solutions or in the presence of water-based solvents or surfactants such as anionic, nonionic, cationic, amphoteric, and zwitterionic surfactants. U.S. Pat. No. 5,723,218 Haugland discloses diffusely dyeing microparticles with one or more dipyrrometheneboron difluoride dyes by using a process, which is essentially similar to the Bangs method. However, when beads internally stained with two separate dipyrrometheneboron dyes, were excited at 490 nm wavelength, they exhibited overlapping emission spectra. Hence, the beads were monochromatic and not multicolored. U.S. Pat. No. 5,326,692 Brinkley et al; U.S. Pat. No. 5,716,855 Lerner et al; and U.S. Pat. No. 5,573,909 Singer et al. disclose fluorescent staining of microparticles with two or more fluorescent dyes. However, dyes used in these processes have overlapping excitation and emission spectra allowing energy transfer from the first excited dye to the next dye and through a series of dyes resulting in emission of light from the last dye in the series. This process was intended to create an extended Stokes shift, i.e., a larger gap between the excitation and emission wavelength, and not the emission of fluorescence from each dye simultaneously. Thus, due to various reasons such as dye-dye interaction, overlapping spectra, non-Gaussian emission profiles and energy transfer between neighboring dyes, the demand for multicolored beads simultaneously emitting fluorescence at distinct peaks was not satisfied.
U.S. Pat. No. 5,786,219 Zhang devised microspheres with two-color fluorescent “rings” or microspheres containing a fluorescent spherical “disk” combined with a fluorescent ring. Nevertheless, such beads, designed for calibration purposes, cannot be used in multiparameter analysis since two dyes were mixed only at one fixed ratio. However, the highest number of dyes ratios ever attempted with at least two dyes never exceeded five.
Chandler et al (U.S. Pat. No. 6,599,331) disclose a method that is essentially similar to that disclosed by Bangs and later applied by Haugland, Brinkley and Lerner. The main difference being the choice of fluorescent dyes. The inventors were able to find a combination of dyes that resulted in dual emission from the particles. However, this method may be limited to a few selected dyes since previous results by Haugland Brinkley and Lerner showed that energy transfer resulted in monochromatic emmission. In U.S. Pat. No. 6,649,414, Chandler et al disclose a method where nano-particles are dyed according to the same procedure as that disclosed in U.S. Pat. No. 6,599,331. These nanoparticles were then attached to the surface of larger polymer particles to generate a new particle consisting of a core particle and a layer of variable numbers of nano-particles on the surface. Such particles will, however, have an irregular surface and therefore highly variable light scattering properties and most likely a high tendency for aggregation in solution. In addition, non-specific binding of proteins from e.g. a cell lysate will tend to increase when the surface is irregular.
The following challenging aspects are relevant when developing a multi-colored particle:
1. Since surface labeling occurs via reactive groups on the particle, binding of fluorescent dyes and capture reagents will compete for the reactive groups on the particle. Thus, particles that are first labeled with different amounts of dyes would not be expected to bind similar levels of reagent used to bind the analyte. Alternatively, particles that have first bound the reagent used to bind the analyte would be expected to have few groups available for the reactive groups of the fluorescent dyes.
2. Surface labeling with multiple fluorescent compounds might be expected to lead to a large degree of fluorescence energy transfer between the dyes. This would greatly limit the number of codes that can be generated. Color-coding based on two or more fluorescent probes implies that the emission and, or absorption spectra of the probes are sufficiently different to allow simultaneous independent detection of the two probes. When two probes are in close proximity fluorescence energy transfer may occur. This implies that the light emitted by one of the probes is absorbed by the second and thus quenched. This phenomenon is well known and may occur even between probes that have large differences in emission and absorption spectra. An example is Phycoerythrin and Cy.5, where the emission spectrum of Phycoerythrin and the absorption spectrum of Cy5 is separated by >100 nm. In this case the fluorescence of Phycoerythrin is completely quenched by Cy5. When dyes are incorporated into the polymer, they are distributed throughout the volume of the particles. The surface area of the particle is a much smaller distribution area for the probes. Therefore one might expect that the probes would be in close proximity. This could limit the number of measurable color codes to the extent that true multiplex color coding would be impossible.
3. It is expected that fluorescent dyes bound to the surface of particles may interfere with fluorescent signals from the analyte due to fluorescence energy transfer. Thus, if the fluorescent probe used to detect the analyte can transfer energy to the dye used for color-coding or vice versa, one would expect that the analyte signal would be different on particles with different color codes.
4. Furthermore, one would expect that surface labeling is not sufficiently stable to allow discrimination of small differences in fluorescence when particles are subjected to storage or reactions that require harsh conditions such as high temperatures.
5. Lastly, fluorescent dyes may undergo changes in spectral characteristics upon binding to monodisperse latex spheres.
In our opinion, no reliable microsphere populations or subsets emitting, upon exposure to a single excitation wavelength, multiple fluorescent signals of variable intensity and at spaced, optically distant wavelengths from surface-bound dyes or a combination of internal and surface-bound dyes have so far still been disclosed. In particular, there is a great need for particles with said characteristics which further permit use of a wide range of commercially available reactive forms of fluorescent dyes, which are produced by a simple and cost-effective method and which can be dyed after labeling with uniform levels of a capture reagent.